JPH04504117A - glycosylated insulin - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] glycosylated insulin The present invention particularly relates to glycosylated insulin and combinations thereof, The present invention relates to pharmaceutical compositions containing the compounds and methods for their preparation.

In recent years, several insulin analogues have been proposed for the treatment of diabetes.

The purpose of developing such insulin analogues is to Available insulins with faster or longer insulin activity The aim is to improve insulin therapy by producing phosphorus analogues.

By substituting one or more amino acid residues in natural insulin, A problem in developing phosphorus analogues is the strong immunogenicity of such compounds. . Also, such substitutions can lead to unpredictable solubility and stability problems. There is a possibility that

Although insulin has an extremely short half-life in the circulating blood, cannot be excluded. In other words, a small amount of insulin is Topics in clinical andexperimental Aspects of diabetes mellitus J ((19 85) +201~20CSakamoto, Min and Bdbd, Eds , + Hercelville Science Publishers B, V, ). It is glycosylated in vivo not only in patients with diabetes but also in non-diabetic patients.

The goal is to develop a structure. Furthermore, the following may also be possible.

That is, the conformational change of the saccharide moiety is to disguise the antigen.

Glucose, mannose and certain oligosaccharides for insulin In the past, the in vivo formation of glycosylated insulin was observed in diabetic patients. The subject in an in vitro study aimed at investigating whether it is the cause of later complications in Met. Biochimica et Biophys ioaActa 386 (1975), 603-607) by the equilibrium dialysis method. The binding of D-glucose to insulin was studied. Binding is highly specific Furthermore, glucose molecules bound to insulin molecules cannot be found in The average number of cases was 8.

The interactions of insulin with glucose and mannose are (Febs Letters 100 (1979), 133-136) investigated. The results shown indicate that both hexoses were incubated in vitro at 37°C. After injection, it was covalently incorporated into the insulin molecule. Selection Under the selected reaction conditions, Glucose for coupling glucose-regulated insulin delivery systems with lectins produced by synthesizing glycosylated insulin derivatives that are comparable to lucose. Brownlee and Ceramics (Science 206 (1979) ), 1190, 1191° and Diabetes 32 (1983), 499 -505). In this study, maltose and other oligos Satucaride reacted with insulin.

The proposal (dowage) is a test of insulin in vitro and in vivo (diabetic patients). studied the non-enzymatic glycosylation of They concluded that it is incorporated into the insulin molecule. In in vitro studies, 3 It has been found that a molecule of glucose is incorporated per molecule of insulin.

1988, Lapolla et al. (Diabetes 37 (1988), 787-791 ) shows that the biological activity of glycosylated insulin in vitro is reduced in vivo. It was reported that According to Torhofel (ibid.), insulin is 37° glycosyl at ambient high glucose concentration at a pH value of 7.4 for 17 h in an aqueous solution of C. be converted into Glucose introduction is an average of 2 moles of glucose per mole of insulin. It turned out to be a source.

Natural insulin contains three free primary amino groups, namely Bl (Phe), Considering that they have AI (Gly) and B29 (Lys), respectively , the above-mentioned glycosylated insulin preparations all contain glycosylated insulin molecules. It would be a heterogeneous mixture of

No one has hitherto taken steps to separate the mixture into its individual components.

Glycosylated insulin inducer for self-regulating insulin delivery system The conductor is as described in Kim et al. (Journal of Control-11ed Re1ea se on (1984), 57-66 and U.S. Pat. No. 4,483.792; 4.4 78.830; 4,478,746 and 4.489.063) has been done. In these glycosylated insulins, glucose or manganese North is a dicarboxylic acid, acid anhydride or phenylamine or a combination thereof It is bound to insulin via a spacer group derived from

European Patent Application No. 84200328 (Publication No. 119.650) is filed by Kim (ibid.) with a spacer group similar to the glycosylated insulin described by (ibid.). Contains galactosyl insulin.

The object of the present invention is to prepare insulin derivatives possessing improved properties. be. More particularly, it is an object of the present invention to develop non-antigenic insulin derivatives. There is a particular thing. Furthermore, it is an object of the present invention to induce insulin faster than natural insulin. Developing insulin derivatives with insulin activity and In order to enable the use of highly concentrated solutions in the pump, The purpose is to improve the solveability. Furthermore, it is an object of the present invention to improve fibrillation. The objective of the present invention is to prepare insulin derivatives with high stability.

The present invention particularly provides glycosylated insulin. Below, especially glycosylation Insulin is an insulin that has carbohydrate substituents at specific positions within the insulin molecule. shall mean Shrine. Surprisingly, such a specifically glycosylated Formulated insulin has certain therapeutic properties, as will be clear from the following description and examples. Provides therapeutic benefits.

In its broadest aspect, the invention provides specifically glycosylated insulin. do. In a narrower aspect, the invention can be applied to positions A, , B or positions AI, BI or is B2. monoglycosylated at; positions tA+ and B+; A1 and and BZ9; or B, and diglycosylated with Fiz’s or triglycosylated at positions AI, Br and B29. Provides a phosphorus derivative.

The glycosylated insulin described by Kim et al. and processed as above was , which is far from the insulin of the present invention, is an artificial spacer group. sugar's own aldehyde to form a covalent bond with insulin without using Use the features. Another advantage of the present invention over the insulin of Kim et al. preservation of the natural charge distribution for the insulin molecule. Therefore, glycosylation The amino group, which is converted into a secondary amino group by reaction (see diagram), is As in phosphorus, it can be protonated.

The insulin derivative of the present invention has monosaccharides or three monosaccharides at each of three positions. It has oligosaccharides with sugar residues up to. A suitable monosaccharide is a group cose, mannose, and galactose. Suitable oligosaccharides are Lutose, isomaltose, lactose, maltotriose, melibiose and and cellobiose.

The specifically glycosylated insulin derivatives of the present invention are useful for the treatment of diabetes. It can be used as For the purpose of monitoring insulin therapy, Selected mixtures of cosylated compounds can also be used.

As used in the present invention, the term "insulin" refers to the human, bovine or porcine Not only does it cover natural forms of insulin such as insulin, but it also covers Natural one or more amino acid residues have been substituted, added, or Also meant is a deficient insulin derivative.

As mentioned above, natural insulin has three possible glycosylation sites, namely A chain and the two N-terminal amino acid residues in the B chain and position B2. Rishi in It has an integer residue. The following is clear. That is, the above type of insulator The number of possible glycosylation sites in a phosphorus homolog depends on how many lysine residues are altered. There are two (two N-terminal residues) to one residue depending on the presence in the insulin molecule. It is more than that. Lysine is the only naturally occurring lysine with a free primary amino group in its side chain. It is a sneaky amino acid.

Glycosylation proceeds schematically with D-glucose according to the following scheme: OH0H D-glucose D-glucose Pyranose structure chain structure 1 Deoxy-D-Fructosyl Insulin (Gly) The above reaction 1 (12 will proceed in a manner.

Specific examples of glycosylated insulin of the present invention are as follows: Phe (Bl) glucose human insulin.

Phe (Bl) Mannose Human Insulin.

cly(＾1)mannose human insulin.

Lys (B29) Mannose Human Insulin.

Phe (Bl) Galactose Human Insulin.

Gly (At) galactose human insulin.

Lys (B29) Galactose Human Insulin.

Phe (Bl) maltose human insulin.

Phe (81) Lactose Human Insulin.

Gly (AI) glucose human insulin.

Gly (AI) maltose human insulin.

GIV (^1) Lactose human insulin.

Lys (B29) Glucose Human Insulin.

Lys (B29) Maltose Human Insulin.

Lys (B29) Lactose Human Insulin.

Gly (AI), Phe (Bl) diglucose human insulin.

Gly (Al), Lys (B29) diglucose human insulin hmm.

Phe (Bl), Lys (B29) diglucose human insulin hmm.

Phe (Bl) isomaltose human insulin.

Guy (AI) Isomaltose Human Insulin.

Lys (B29) isomaltose human insulin.

Phe (Bl) maltotriose human insulin.

Guy (Al) Maltotriose Human Insulin.

Lys (B29) Maltotriose Human Insulin.

Gly (Al), Phe (Bl) Dimaltose Human insulin.

Gly (AI), Lys (B29) Dimaltose Human insulin.

Phe (Bl), Lys (B29) Dimaltose Human insulin .

Gly (Al), Phe (Bl), dilactose, human insulin.

Gly (Al), Lys (B29) dilactose human insulin.

Pbe (Bl), Lys (B29) dilactose human insulin hmm.

Gly (Al), Phe (Bl) dimaltotriose human insulin.

Phe (Bl), Gly (AI) dimannose human insulin.

Phe (Bl), Lys (B29) Dimannose human insulin hmm.

Gly (AI), Lys (B29) dimannose human insulin.

Phe (Bl), Gly (81) digalactose human insulin.

Phe (Bl), Lys (B29) digalactose human insulin Rin.

Gly (Al), Lys (B29) digalactose human insulin.

Phe (Bl), Gly (Al) diisomaltose human insulin.

Phe (Bl), Lys (B29) diisomaltose human inci Urin.

Guy (81), Lys (B29) diisomaltose human inci Urin.

Phe (Bl) Glucose [ASp110] Human insulin.

Gly (Al), Phe (Bl) Glucose [ASpl IO] Human In Shrine.

Also of interest are specifically glycosylated insulins from other species, such as pigs. be.

The glycosylated insulin of the present invention is different from insulin or insulin homologues. an excess of the selected monosaccharide or oligosaccharide and a suitable organic or aqueous It can be prepared by reaction in a neutral medium. Temperatures range from 20″C to 6 It can vary within a range of 0°C. Lower carboxylic acids such as acetic acid, etc. and propionic acid, lower aliphatic alcohols such as methanol, ethanol and and 2-propanol, ethylene glycol and propylene glycol can be used. Wear. However, phenol can also be used.

Reaction times and reaction mixture compositions vary depending on mono-, di- or tri-glycosylation. It depends on the desired product.

The reaction is conveniently carried out by reverse phase high performance liquid chromatography (hereinafter referred to as RP HPLC). ) to determine the point of maximum formation of each of the individual glycosylation products. can do.

The reaction is terminated, for example, by cooling to -20°C, and the reaction mixture is concentrated to dryness. Afterwards, the main components of the reaction mixture are isolated and purified by preparative RP HPLC. Ru. After desalting, the product was subjected to fast atom bombardment mass spectrometry (hereinafter abbreviated as FAB-MS), Quantitative amino acid analysis and bioassay after boron hydride (porohydride) reduction Analyze by.

The glycosylated insulin and mixtures thereof of the present invention are novel to those skilled in the art. For human or pig insulin in insulin preparations known as Replaced to give a new insulin formulation. These new insulin preparations , glycosylated insulin or pharmaceutical thereof in an aqueous solution, preferably at an aqueous pH value. Contains acceptable salts. Preferably, the aqueous medium contains zinc ions, buffer compositions, acetate or phosphate and a preservative such as m-cresol. , methylparaben or phenol.

The OpH value of the formulation can be adjusted to the desired value and the formulation can be sterilized by mouth.

The insulin preparation of the present invention can be used in the same manner as known insulin preparations.

experimental part Example I Phe (Bl) glucose human insulin human insulin (0,1 mmo 1) is suspended in methanol (30%) and glacial acetic acid is added at room temperature. Insert the mixture Stir gently until the surin is dissolved. Then, add a certain amount of methanol ( 35d) and after adding D-glucose (2.2 mmol), the mixture was heated to 40° C. for 8 hours, whereby the title compound became the main component.

The solution was concentrated on a rotary evaporator to almost dryness. Dissolve the residue in water and prepare PRH Fractionation was performed by PLC. Column: 16X250ns, 7. mloo human C4 particles.

Temperature 30"C, mobile phase A: 0.04M phosphoric acid, 0.2M sodium sulfate, 1 0% acetonitrile, pH value adjusted to 2.5 with ethanolamine, B: 50% Acetonitrile.

Fractions corresponding to the central part of the main peak were desalted and lyophilized.

The yield was 0.02 mmol. The product was analyzed by FAB-MS and borohydride. It was analyzed and characterized by quantitative amino analysis after reduction. For amino acid analysis, Phe Two phenylalanine residues showing substitutions in (Bl) (i.e. human protein demonstrated the presence of one less phenylalanine residue compared to surin. molecule The amount was found to be 5970 (theoretical value: 5970).

Example 2 Ph, e (Bl), Gly (Al) diglucose human insulin The above compound was prepared similarly as described in Example 1. However, the reaction time is 16 hours. This made the title compound the main component.

The yield was 5 mmol of Q.

Amino acid analysis reveals substitutions at position A, and one less Phe at B+. and the presence of one less Gly residue.

The measured molecular weight was 6132 (theoretical value: 6132).

Essentially, Jorgen et al., Diabetologia 19 (1980) +546-554) using the iodate method described by 125I labeled Phe (Bl), Gly (Al) diglucose insulin The drug was injected into pigs and subcutaneous absorption was measured. 12sl human insulin and 12J Phe (Bl), Gly (Al) diglucose human insulin subcutaneously administered to pigs The absorption rate after injection is shown in Table 1. T in Table 1? 5. T.S. and T’zs value The radioactivity measured at the injection site from the time of sample injection was 75%, 50%, and 50%, respectively. From Table 1, it is the time (in hours) that elapsed until the glycosylation decreased by 25%. Insulin is absorbed much faster than human insulin.

Table 1 Human insulin 1.12 2.33 3.69 diglyose human insulin Human insulin 0.65 1.47 2.76 Human insulin (Acrapid (registered) Recording surface [[) and Gly (Al), Phe (Bl) diglucose human insulin 0. Human insulin by subcutaneous injection into pigs in the amount of IU/)cg The blood glucose lowering effect of (average value of 5 animals) is shown in Table 2 below. Second In the table, the value for glucose (nmol/j!) is given.

l grass -0,335,305,24 05,365,32 0,334,784,76 0,674,724,06 14,123,34 1,53,602,98 23,242,84 2,53,282,96 3'3.18 3.14 4 3.34 3.96 Table 2 shows the rapid action of diglucose insulin compared to human insulin. Indicates purpose.

Human insulin, bovine insulin and Gly (Al), Phe (Bl) Immune response to diglucose human insulin in domestic rabbits (for 10 animals) The average value) is shown in Table 3 and shows the value for % binding in rabbit serum (method difference). Yuri et al. (Horm, Metab, Res, 5upp1. Ser, 5 (1974) ), 134-143)).

“Man” L table 0 1.9 -0.4 1.2 13 2.3 0.6 1.3 27 3.0 2B, 5 1.5 41 3.2 29.7 1.4 55 4.3 30.7 1.1 69 3.9 32.7 2.2 83 2.4 30.1 1.1 97 2.0 32.6 1.5 As is clear from Table 3, diglycose human insulin is have a very low immune response in response to human immune responses.

Example 3 The following compounds were prepared similarly. Measured by FAB-MS or plasma desorption mass spectrometry The determined and calculated molecular weights are shown below for each compound.

j-1 Phe (Bl) Galactose Human Insulin 5956 5970Ph e(Bl) Maltose Human insulin 6119 6132Phe(B l) Lactose Human Insulin 6125 6132Phe (Bl) ? Lutotriose human insulin 6288 6294Gly (AI), Phe (Bl) Simaltose Human Insulin 6444 6456Gl y (AI), Phe (Bl) dilactose human insulin 6446 6456Gly (Al), Phe (Bl) dimaltotriose human inci Urin 6771 6780Gly (AI), Phe (Bl), Lys (B29 )) Liglucose Human Insulin 6294 6294Gly (AI) , Phe (Bl) Digurgose [AspII°] Human Insulin 6 112 6110 The position of the linked sugar residue is determined by polyhydride reduction and quantitative amino acid Confirmed by analysis.

international search report international search report

Claims (15)

[Claims] 1. one or more monosaccharide groups or one or more monosaccharide groups with up to 3 sugar units is a specifically glycosylated infusion containing more oligosaccharide groups. Shrine. 2. 1 monosaccharide group or 1 oligosaccharide with up to 3 sugar units The glycosylated insulin according to claim 1, which contains a lido group. 3. 2 monosaccharide groups or 2 oligosaccharides with up to 3 sugar units The glycosylated insulin according to claim 1, which contains a lido group. 4. 3 monosaccharide groups or 3 oligosaccharides with up to 3 sugar units The glycosylated insulin according to claim 1, which contains a lido group. 5. Claim 2, which is monoglycosylated at position A1, B1 or B29. Glycosylated insulin as described in Section. 6. positions A1 and B1: A1 and B29; or diglyco at B1 and B29 The glycosylated insulin according to claim 3, which is sylated. 7. Claim No. 3, which is triglycosylated at positions A1, B1 and B29. The glycosylated insulin according to item 4. 8. The group according to any one of claims 1 to 7, containing Asp at position B10. Lycosylated insulin. 9. Claims 1 to 8, wherein the parent insulin species is human. Glycosylated insulin. 10. Phe (B1) glucose human insulin. 11. Phe (B1), Gly (A1) diglucose human insulin. 12. at least 90%, preferably at least 95%, most preferably less Specifically glycosylated according to any one of claims 1 to 11 with 99% A composition containing insulin. 13. Specifically glycosylated according to any of claims 1 to 12 Insulin or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable salt thereof. Pharmaceutical formulations containing adjuvants and additives and preservatives acceptable as auxiliaries and additives and preservatives. 14. Specifically glycosylated according to any of claims 1 to 13 A method for producing insulin, which comprises adding suitable insulin to an aqueous medium or an organic medium. Monosaccharides with up to 3 sugar units and free aldehydes in a solvent or oligosaccharides with up to 3 sugar units and free aldehydes The above-mentioned method of production comprises reacting the target product and then isolating the desired product from the reaction mixture. 15. Novel features or combinations thereof described herein.